Fluorescence Digital Image Gallery

Embryonic Swiss Mouse Fibroblast Cells (3T3)

The history of synthetic fluorescent probes dates back over a century to the late 1800s when many of the cornerstone dyes for modern histology were developed. Among these were pararosaniline, methyl violet, malachite green, safranin O, methylene blue, and numerous azo (nitrogen) dyes, such as Bismarck brown. Although these dyes were highly colored and capable of absorbing selected bands of visible light, most were only weakly fluorescent and would not be useful for the fluorescence microscopes that would be developed several decades later. However, several synthetic dye classes synthesized during this period, based on the xanthene and acridine heterocyclic ring systems, proved to be highly fluorescent and provided a foundation for the development of modern synthetic fluorescent probes. Most notable among these early fluorescent dyes were the substituted xanthenes, fluorescein and rhodamine B, and the biaminated acridine derivative, acridine orange.

A monolayer culture of Swiss mouse embryo cells (illustrated above) was immunofluorescently labeled with primary mouse anti-alpha-tubulin antibodies, and then subsequently treated with a mixture of secondary antibodies conjugated to Alexa Fluor 568 in a mixture containing phalloidin conjugated to Alexa Fluor 350. The cell nuclei were counterstained with SYTOX Green. Images were recorded in grayscale with a QImaging Retiga Fast-EXi camera system coupled to an Olympus BX-51 microscope equipped with bandpass emission fluorescence filter optical blocks provided by Omega Optical. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.